This invention relates to a method of diagnostic imaging of a human or animal subject, in particular a method in which the image is generated from detected light generated by or characteristically affected by ultrasound irradiation of the subject.
Optical imaging, also called light imaging, is perhaps one of the oldest of medical tools for screening and diagnosis. However, even now, optical imaging is largely limited to body surfaces. The primary advances in optical imaging have served essentially only to expand the range of body surfaces accessible to optical imaging techniques. Thus for example endoscopic techniques have made it possible to image from within the gastrointestinal tract, the cardiovascular system, the bladder, the vagina and uterus as well as the external surface of almost any internal organ. However, the deep interior of most organs, several centimeters beneath the surface, remains almost inaccessible to optical imaging.
There are two primary difficulties associated with subsurface optical imaging. These arise from light absorption and light scattering.
Naturally occurring substances in the body strongly absorb most visible light before it has travelled through typical tissue to any significant extent. Thus by way of example the absorption coefficient for light of 515 nm wavelength by human liver is 18.9 cmxe2x88x921 which means that on average a photon at 515 nm travels only about 0.5 mm in the liver before it is absorbed.
Because the photon follows a non-linear path within the tissue as a result of scattering, the actual depth of penetration before absorption is less than the pathlength of 0.5 mm.
Fortunately there is a wavelength xe2x80x9cwindowxe2x80x9d at 600 to 1300 nm in the red to near infrared region in which light absorption by the body is relatively weak. Thus for liver and breast tissue the absorption coefficients for light of wavelength 635 nm are only 2.3 cmxe2x88x921 and 0.2 cmxe2x88x921 giving mean pathlengths before absorption for photons at 635 nm of about 4.3 mm and 50 mm respectively. At these wavelengths, bone and brain are also relatively transparent so that absorption alone is not a barrier to light imaging with red to near infrared light. Thus, the penetration depth of light at 635 nm is an order of magnitude greater than that of light at 515 nm.
Light scattering however remains a major obstacle to light imaging of subsurface structures. Thus while light of wavelengths 600 to 1300 nm may pass through tissues and organs, the scattering that occurs means that the information on subsurface structures that would be extractable from the detected transmitted or reflected light is largely lost and small or deeply buried structures are not detectable distinctly by eye. At 635 nm, the scattering coefficient for human breast tissue is 395 cmxe2x88x921 meaning that while on average a photon will travel several centimeters before being absorbed it is constantly diverted by scattering events which occur on average every 2 xcexcm. In other tissues, the scattering may be less severe but it is still substantial. The typical photon will travel only 16 xcexcm between scattering events in brain grey matter for example.
While light scattering in the body is random, it is also highly anisotropicxe2x80x94the paths of the photon before and after a scattering event are not on average highly divergent. This scattering type is typical of Mie scattering.
To take account of the anisotropy of scattering, the reduced scattering coefficient xcexcsxe2x80x2 is used in place of the simple scattering coefficient xcexcs in certain mathematical models. xcexcsxe2x80x2 is related to xcexcs by the equation xcexcsxe2x80x2=xcexcs(1xe2x88x92g) where g is the average cosine of the angle between the photon""s incoming and departing paths for scattering events. For human grey matter xcexcsxe2x80x2 is only 7.22 cmxe2x88x921 meaning that light travels about 0.1 mm before its direction of propagation is significantly altered. This means that even after passing through several centimeters of breast or other tissue a light beam may still have a significant component which is travelling in substantially the same direction as the incident light beam. This component is often referred to as the quasi-ballistic component and it is this component which is of particular utility in light imaging of subsurface structures.
Thus, since the flight path of the quasi-ballistic photons through tissue is shorter than the path of the more highly scattered photons, the diffuse component, it is possible to separate out the transmitted light into its quasi-ballistic and diffuse components. This may be done for example by using a pulsed light source and detecting the leading edges of the transmitted pulsed light.
Despite its technical difficulties, light imaging has important advantages over other medical imaging modalities in that it can provide functional information as well as spatial information about the body. Thus with suitable modification it may be used for example to measure pH, oxygen content, metal concentration, etc.
Acousto-optical imaging is a modified approach to light imaging in which focused ultrasound is used to isolate optical signals from the body. Several mechanisms of interaction are possible. In one of these the acoustic wave sets up moving regions of different pressure, density and refractive index that interact with the light in much the same way as a diffusion grating. The movement of the sound waves moreover induces a Doppler shift of the sound frequency into the light frequency making it possible to identify that portion of the light that has actually interacted with the sound wave. Thus the light that has passed through the focused ultrasound region may be separated from other components of the detected light because it is shifted in frequency and wavelength. Acousto-optic imaging is described for example in U.S. Pat. No. 5,171,298, and by Wang et al. Optics Letters 20: 629-631 (1995), Wang et al. Proc. Opt. Soc. Amer. ATuB3-1: 166-168 (1996), and Brooksby et al. Proc. Soc. Photo-Opt. Instr. Engin. 2389: 564-570 (1995).
Acousto-optic imaging actually expands the ability of light imaging to provide functional imaging since the degree to which the focused sound waves interact with the light will depend upon the mechanical properties of the body at the focus site. The ability to measure tensile modulus and other mechanical properties of a suspicious lesion greatly facilitates identification of the lesion as malignant or benign.
Thus, in acousto-optic imaging the detected light signal carries a record of the interaction of ultrasound on the test object. Other phenomena also have this characteristic. In the phenomenon known as sonoluminescence, light is generated by the action of ultrasound on certain materials (see Suslick, xe2x80x9cUltrasound, its chemical, physical and biological effectsxe2x80x9d, VCH, New York 1988). Modulation of the frequency or amplitude of the ultrasound may impart a modulation to the sonoluminescence, and detection at the modulation frequency provides a means of separating out from the background the signal due to the ultrasound irradiation.
The present invention is directed at improvements in light imaging procedures where the detected light signal is affected by ultrasound irradiation of the human or animal body under study and in particular to the use of contrast agents in such imaging procedures. These contrast agents are materials which scatter, emit or absorb (or do two or more of these) light in the 300 to 1300 nm wavelength range, preferably the 600 to 1300 nm wavelength range, whereby the detected light signal is affected or generated by the ultrasound irradiation of the body, by the presence in (or absence from) the ultrasound irradiated portion of the body of the contrast agent and optionally by the selective sensitivity of the contrast agent to differences in its microenvironment within the body.
Thus viewed from one aspect the invention provides a method of generating information from (e.g. an image of) an animate human or non-human (e.g. mammalian; avian or reptilian) animal body which method comprises:
administering to said body a physiologically tolerable material capable of absorbing, scattering or emitting light at a wavelength in the range 300 to 1300 nm;
subjecting at least a portion (or target zone) of said body to ultrasound irradiation;
detecting light in the wavelength range 300 to 1300 nm, preferably 600 to 1300 nm, from said portion of said body; and
manipulating the detected light to generate said information (e.g. an image of at least part of said body).
By this method it is possible to to modify or enhance said information (e.g. to enhance contrast in an image of said portion or to facilitate determination of functional information regarding said portion, such as pH or oxygen content, etc.).
Viewed from an alternative aspect the invention provides a method of generating information from (e.g. an image of) an animate human or non-human (e.g. mammalian, avian or reptilian) animal body which method comprises:
administering to said body a physiologically tolerable material capable of modifying the generation or transformation of light within said body by ultrasound irradiation;
subjecting at least a portion of said body to said ultrasound irradiation;
detecting light from said portion of said body; and
manipulating the detected light to generate said information (e.g. an image of at least part of said body).
The modifying effect of the said material is preferably an enhancing effect and the light detected and used for information generation preferably comprises ultrasound induced light.
The physiologically tolerable material (hereinafter referred to as the contrast agent) used in the methods of the invention may conveniently be one for which the contrast effect, e.g. the effect on light generation, light scattering, light absorption, light propagation or light frequency is dependent on microenvironment, e.g. pH, oxygen content, etc.
Viewed from a further aspect the invention provides the use of a physiologically tolerable material capable of absorbing, scattering or emitting light at a wavelength in the range 300 to 1300 nm, preferably 600 to 1300 nm for the manufacture of a composition for administration to the human or animal body in a method of diagnosis practised thereon which involves subjecting a portion of said body to ultrasound irradiation, detecting of light in the wavelength range 300 to 1300 nm, preferably 600 to 1300 nm from said portion and manipulation of the detected light to provide spatial and/or functional information (e.g. an image) relating to said portion.
Viewed from a still further aspect the invention provides the use of a physiologically tolerable material capable of modifying light generation or transformation within a human or animal body for the manufacture of a composition for administration to the human or animal body in a method of diagnosis practised thereon which involves subjecting a portion of said body to ultrasound irradiation, detecting of light from said portion and manipulation of the detected light to provide spatial and/or functional information (e.g. an image) relating to said portion.
In the methods of the invention, the ultrasound irradiation is preferably focused on a portion of the body to which the contrast agent distributes or at which the contrast agent has accumulated. The ultrasound irradiation moreover is preferably modulated, in frequency and/or amplitude, with a characteristic modulation pattern or frequency and the component of the detected light signal that is likewise modulated is preferably extracted and used to generate the desired information (e.g. image).
Where the contrast agent is not itself sonoluminescent, the method of the invention will generally involve exposing at least part of the ultrasound irradiated portion (the target zone) of the body with light having a wavelength in the range 300 to 1300 nm, preferably 600 to 1300 nm, preferably monochromatic, e.g. laser, light. Such irradiating light incident on the body may moreover be frequency and/or amplitude modulated. As one example of amplitude modulation, the incident light may be pulsed.
Where the body is thus illuminated, the detected light will generally correspond to a transmitted or scattered (e.g. reflected) component of the illuminating light or to light emitted by the contrast agent following absorption of the illuminating light (e.g. a fluorescence emission).
Where the body is illuminated, this may be with one or more, e.g. 1, 2, 3 or 4, non co-axial beams of light. Where a plurality of light beams are used, these may be at different wavelengths although generally it will be preferred to use beams of the same wavelengths. Moreover when a plurality of light beams are used these will generally be directed at the target zone and means will generally be provided for detecting light from each light beam that has passed through the target zone. Each beam may be amplitude- or frequency-modulated at the same frequency or at different frequencies. When the light is highly scattered in the body, the direction of illumination is arbitrary.
The contrast agent used for acousto-optic imaging according to the invention may take a variety of forms. Thus it may be a particulate (e.g. a solid or semi solid particle, a liquid droplet, a gas bubble, or a vesicle (e.g. a micelle, liposome or microballoon) with a rigid or flexible, continuous or porous membrane enclosing a gas, a gas-precursor (a material or mixture of materials which are gaseous at 37xc2x0 C. or at temperatures generated by the ultrasound irradiation), a liquid, or a solid, or a mixture of two or more thereof.
For the sake of clarity, the word xe2x80x9cparticlexe2x80x9d is used to refer to any physiologically acceptable particulate materials. Such particles may be solid (e.g. coated or uncoated crystalline materials) or fluid (e.g. liquid particles in an emulsion) or may be aggregates (e.g. fluid containing liposomes). Particulate material with a particle size smaller than or similar to the incident light wavelength is preferred.
Such particulates may be or include chromophores (materials or moieties which absorb and/or emit light at a wavelength in the range 300 to 1300 nm, preferably at a wavelength in the range 600 to 1300 nm) or may be essentially colourless (e.g. free from prominent absorption or emission maxima in the wavelength range 600 to 1300 nm). Thus for example particulate contrast agents may be materials which scatter light in the 300 to 1300 nm, preferably 600 to 1300 nm wavelength range by virtue of their size and difference in refractive index from that of surrounding body fluids in the target zone. Alternatively they may be composed of a light-absorbing dye with or without a colourless surrounding shell or membrane, or of a colourless core (solid, liquid or gaseous) surrounded by or coated with a light-absorbing dye. For sonoluminescence, particles with chromophores are particularly useful if they absorb light in a wavelength range outside the detection range. Where particulate, the contrast agent will preferably have a mean particle size in the range 5 nm to 20 xcexcm (the upper end of the range generally being appropriate only for deformable particles), particularly 10 nm to 8 xcexcm, especially 80 to 2000 nm and most preferably 100 to 500 nm.
Generally, lipophilic contrast agents will be formulated as oil-in-water emulsions with oil droplet sizes between 5 and 10000 nm, preferably between 10 and 2000 nm suspended in a pharmaceutically acceptable aqueous phase. Such oil droplets may be composed exclusively of radiation absorbing, scattering or fluorescing component(s) or may include other lipophilic substances distributed throughout the droplet. These emulsions will likely contain pharmaceutically acceptable excipients as are known in the art including lecithin, other phospholipids, surfactants such as the Tetronics and Pluronics, lipophilic additives such as sesame oil, and normally used components for isotonicity, pH and osmolality control.
The contrast agent may alternatively be a soluble material, e.g. a water-soluble chromophore or polychromophore, or a colourless soluble polymer which facilitates the generation of light by ultrasound.
Where the contrast agent is a, or contains at least one, chromophore, this is preferably a material or moiety which has an absorption maximum in the range 300 to 1300 nm, preferably 600 to 1300 nm, particularly preferably 650 to 900 nm.
For fluorophores, i.e. materials which absorb light and emit light at a different wavelength, the emission wavelength will preferably also be in the 600-1300 nm range. In this way the light the chromophore absorbs/emits is subject to minimal absorption by the body.
Where the contrast agent contains a chromophore, it may conveniently be one which has characteristic absorption/emission maxima which are sensitive to the micro-environment the contrast agent is in, e.g. pH, oxygen content etc. For particulate chromophore-free contrast agents, sensitivity to microenvironment may likewise be achieved by forming the particles from materials which change optical or mechanical properties (e.g. hardness, opacity, size etc.) according to the pH, oxygen content etc. of their microenvironment.
Thus the absorption wavelengths and acousto-optical properties of the contrast agent can be selected to be sensitive to the biochemical or biophysical properties of the organ or tissue to which they distribute enabling information concerning those biochemical or biophysical properties to be extracted from the light signals detected in the method of the invention.
The xe2x80x9cinformationxe2x80x9d generated in the method of the invention may be in the form of spatial, temporal and/or functional images, in the form of quantitated values for selected parameters for the target zone (e.g. pH, etc.), or simply in terms of an indication that a criterion is or is not met (e.g. pH is or is not different from that of surrounding tissue). Where quantitated values are generated, this will generally involve calibration against values for known standards.
Particulate or soluble contrast agents used according to the invention may if desired incorporate components serving to modify their biodistribution or bioelimination, e.g. targeting vectors (e.g. antibodies, antibody fragments, proteins, oligopeptides, receptor binding drugs, etc.) capable of causing the agent to accumulate at particular body sites, for example in tumors or at the surfaces of body ducts or cavities (e.g. in the gastrointestinal tract, lungs, vasculature etc.), or blood pool residence extenders (for example polyalkylene oxides, heparinoids and other materials which can delay abstraction from the blood stream of particulate or soluble materials). Examples of such vectors and blood pool residence extenders and their conjugation to particles and chromophores are given in our copending US Patent Application entitled xe2x80x9cMethod of Tumor Treatmentxe2x80x9d filed Apr. 29, 1997 and our copending International Patent Application No. PCT/GB98/01245 entitled xe2x80x9cMethod of Demarcating Tissuexe2x80x9d, which also list suitable chromophore and polychromophore materials.
In general, solid contrast agents will be formulated as particles with sizes between 5 and 10000 nm, preferably 10 and 6000 nm, suspended in aqueous solution. However, optimal light absorption and optimal light scattering will occur when the particles have diameters between 100 and 500 nm.
Optionally the solution in which solid particles are suspended may contain buffers and other excipients to control the pH and osmolality. Preferably the solid particles will be coated with a surfactant selected from, e.g. Pluronic F-68, Pluronic F-108, Tweens, Spans, and Tetronic T-908, to impede aggregation during autoclaving and storage.
In this invention, a surfactant molecule is defined as an emulsifier or detergent as listed in McCutcheon""s Directories, Volume 1: Emulsifiers and Detergents (1994), and which contains at least one chemical functional group selected from the group consisting of an alcohol (OH), a nitrilo group including a primary amine (NH2) and a secondary amine (NH), a carboxylic acid (COOH), a sulfhydryl (SH), a phosphoric acid group, phosphonic acid group, a phenolic group, a sulfonic acid group, a carbon-carbon double bond, and a ketone.
Chemical functional groups in the surfactant molecules can be interconverted by chemical reactions well known to those skilled in the art. For example, a hydroxyl group can be converted to a methanesulfonic acid ester which can be treated with sodium azide and reduced to form an amine group. Carboxylic acid groups and ketones can be reduced to form alcohols, and alcohols can be oxidized to form ketones, aldehydes, and carboxylic acid groups.
Useful surfactant molecules are emulsifiers or detergents which can function as dispersing agents, wetting agents, adsorbents, anticaking agents, soil antiredispositioning agents, antistats, binders, carriers, pearlescents, conditioning agents, hydrotropes, defoamers, emollients, flocculants, humectants, lubricants, opacifiers, plasticizers, preservatives, release agents, scale inhibitors, stabilizers, suspending agents, thickeners, UV absorbers, water repellants, waxes, and polishes, and which contain at least one chemical functional group selected from the group consisting of an alcohol (OH), a nitrilo group including a primary amine (NH2) and a secondary amine (NH), a carboxylic acid (COOH), a sulfhydryl (SH), a phosphoric acid group, a phosphonic acid group, a phenolic group, a sulfonic acid group, a carbon-carbon double bond, and a ketone.
Preferably, the surfactant molecule comprises a polyalkyleneoxide moiety, optionally containing a branching group as defined herein; more preferably a polyalkyleneoxide block copolymeric moiety, optionally containing a branching group as defined herein; and most preferably a polyalkyleneoxide block copolymeric moiety optionally containing a branching group as defined herein and comprising a polypropylene oxide block and a polyethyleneoxide block. Examples of useful surfactant molecules include block copolymers such as AL 2070 available from ICI Surfactants, Antarox block copolymers available from Rhone-Poulenc, Delonic block copolymers available from DeForest, Inc., Hartopol block copolymers available from Texaco Chemical Canada, Macol block copolymers available from PPG Industries, Marlox block copolymers available from Huls America, Pluronic block copolymers including Pluronic F, L, P and R available from BASF Corp., Poly-Tergent block copolymers available from Olin Corp., and Tetronic and Tetronic R block copolymers available from BASF Corp. Currently preferred surfactant molecules include Tetronic and Pluronic block copolymers, and currently most preferred are Tetronic block copolymers.
When the agents are intended for injection into the vascular system, they may be coated with a substance such as poly(ethylene glycol) to slow clearance from the bloodstream.
Optionally, the surfactants and other compounds of this invention can be attached to a targeting vector so as to allow the particles to accumulate in certain locations of the body such as specific organs, parts of organs or diseased tissue. Methods for attachment are taught in WO 96/40285, its priority applications U.S. Ser. Nos. 08/497,684 and 08/640,464, and in 08/392,614.
Soluble dyes will be useful as contrast agents for acousto-optic imaging because they intensify the interaction between the light and the sound by increasing the difference in optical properties between the troughs and valleys of the acoustic wave. Such dyes will be especially useful if they are highly fluorescent. When the frequency of the ultrasound is properly chosen, it will modify the local environment around the dye and affect the fluorescence frequency. The ultrasound frequency should be between 0.01 and 10 MHz, especially 0.1 to 3 MHz, particularly 0.5 to 2.5 MHz. Modulation of the acoustic power at a frequency between 0 and 100 kHz, especially 0 and 30 kHz, particularly 0 and 10 kHz, will impart an amplitude modulation on the detected light at the same frequency. In addition, the detected light will be modulated at the frequency of the ultrasound. Detection of the light that is modulated at either frequency will facilitate separation of the signal carrying imaging or functional information from the background signal, which largely consists of scattered light from the impinging radiation.
Especially preferred as soluble dyes are polymeric substances with a molecular weight high enough to slow the clearance of the dyes from the blood stream. Such materials may consist of chromophores with attached polymer segments, or they may be substances in which the dyes are directly incorporated into the polymer backbone. Especially preferred for a linker between or for conjugation to dye chromophores are segments of poly(ethylene glycol). Examples of such dyes are disclosed in W097/13490 and in our copending US Patent Application entitled xe2x80x9cMethod of Tumor Treatmentxe2x80x9d filed Apr. 29, 1997 and our copending International Patent Application No. PCT/GB98/01245 entitled xe2x80x9cMethod of Demarcating Tissuexe2x80x9d filed Apr. 29, 1998.
When soluble dyes are to be supplied as pre-made solutions, the solutions optionally may contain stabilizing agents as taught in WO 94/23646. The solutions may also contain excipients to control the pH or osmolality.
Soluble dyes may optionally be enclosed in vesicles (e.g. micelles or liposomes) as taught in WO 96/23424. Liposomal formulations may optionally contain substances to stabilize the dyes against oxidation or other degradative processes.
A preferred form of liposomal formulation contains dyes having in common with rhodamine 6G (Aiuchi, T.; Tanabe, H.; Kurihara, K.; Kobatake, Y., xe2x80x9cFluorescence Changes of Rhodamine 6G Associated with Chemotactic Responses in Tetrahymeena Pyriformis,xe2x80x9d Biochem. Biophys. Acta, 1980, 628, 355-364) and carboxyfluorescein (Chen, R. F.; Knutson, J. R., xe2x80x9cMechanism of Fluorescence Concentration Quenching of Carboxyfluorescein in Liposomes: Energy Transfer to Nonfluorescent Dimers,xe2x80x9d 1988, 172, 61-77) the property that they lose their fluorescence when encapsulated in liposomes. A standard method of preparation of liposomes of uniform size involves sonication. Under the influence of ultrasound the liposomes break and reform. In the body the effect of the focused ultrasound will be to release dye selectively at the focusing site. The detection of fluorescence from that site then pinpoints its location.
Liposomes that release fluorescent dye will be particularly useful as contrast agents when the amplitude of the ultrasound has a frequency between 0.01 and 10 MHz, especially 0.1 to 3 MHz, particularly 0.5 to 2.5 MHz, and is modulated at a characteristic frequency between 0 and 100 kHz, especially 0 to 30 kHz, particularly 0 to 10 kHz. Then the component of the detected signal that directly reflects the location and properties of the site of sound focusing will be also modulated at that frequency and can easily be separated from background. The most effective modulation frequency will be chosen to reflect the rate at which dye is released and recaptured from the liposomes.
Contrast agents for acousto-optic imaging may also have the form of gas-filled bubbles in which dyes having in common with rhodamine 6G and carboxyfluorescein the property that they lose their fluorescence when encapsulated in liposomes are incorporated into the shells. Except for the presence of the dye, these bubbles will have a form similar to that of contrast agents for ultrasound imaging. Heating of the gas contained within these bubbles by ultrasound will lead to expansion of the gas, rupture of the bubbles, and release of the fluorescent dyes. Again, these agents may contain agents on the surface such as PEG to slow blood clearance or may have attached specific targeting vectors.
Sonoluminescence is associated experimentally with the collapse of microbubbles produced by high-intensity ultrasound (cavitation) (Suslick, K. S., ed., xe2x80x9cUltrasound, Its Chemical, Physical, and Biological Effects,xe2x80x9d VCH, New York, 1988). Bubble formation is facilitated by solid particles acting as nucleation centers. Contrast agents consisting of suspended particles with diameters between 200 and 5000 nm will be particularly useful as contrast agents for sonoluminescence. These should be as large as possible within the biological constraints necessary to prevent adverse reactions. In general, the suspended particles will have substances such as poly(ethylene glycol) on their surfaces to slow uptake by macrophages within the body and to prolong blood lifetime. Optionally they may contain specific targeting vectors. Optionally they may also have attached materials to scavenge free radicals that are produced by the cavitation process.
Alternatively contrast agents without radical scavengers will be useful for monitoring therapeutic procedures in which targeted ultrasound is used to destroy diseased lesions.
Cavitation is known to result in the formation of free radicals. When these radicals interact with precursors to form colored free radicals, they can be used for the generation of markers of the site of ultrasound focusing that can detected by optical imaging and used as an indication of where the ultrasound is concentrated. This method will be especially useful when the ultrasound is used for therapeutic purposes, for example, the selective destruction of cancerous lesions, as well as for imaging.
Water soluble polymers will facilitate the generation of light by ultrasound. The water soluble polymers used according to the invention conveniently have a molecular weight (MW) of 150 to 1000000 (especially 500 to 500000, most preferably 1 to 50000), and preferably are hexamers or higher polymers. The polymers preferably contain monomer residues contributing 2 to 6 atoms to the polymer backbone, especially 2, 3 or 4 atoms. The polymers may conveniently comprise residues of monomers such as alkylene oxides, hydroxyalkyl-acrylates or methacrylates, vinyl alcohol, vinyl pyrrolidone, acrylamide, styrenes, etc. Especially preferably, the polymers will be polymeric surfactants.
Examples of suitable polymers include: polyalkylene oxide polymers and copolymers (including random and block and graft copolymers) and oligomers such as poly(ethylene oxide) also known as poly(ethylene glycol) also known as PEG, as well as poloxamers and poloxamines (also known as Pluronics and Tetronics); PEG derivatives such as PEG mono- and bis-ethers of alkyl, alkenyl and alkynyl groups containing from 1 to about 26 carbon atoms and which can be linear or branched and which can comprise a cycloalkyl or cycloalkenyl group with ring size of from 3 to 10 carbons (preferably a cyclohexenyl, cyclooctenyl, or cyclooctadienyl group) such that this total number of carbons in the group is less than 26; PEG mono- and bis-esters (including alpha-methoxy-PEG monoesters) and PEG mono- and bis-amides (including alpha-methoxy-PEG monoamides) of alkyl, alkenyl and alkynyl carboxylic acid groups containing from 1 to about 26 carbon atoms and which can be linear or branched and which can comprise a cycloalkyl or cycloalkenyl group with ring size of from 3 to 10 carbons (preferably a cyclohexenyl, cyclooctenyl or cyclooctadienyl group) such that the total number of carbons in the group is less than 26; and derivatives of PEG as described above conjugated to a polyiodinated aromatic compound, e.g. PEG esters and amides of mono, di, and tri-iodinated aromatic benzoic acid derivatives such as diatrizoic acid esters and amides; poly(propylene glycol) (PPG, also known as poly(propylene oxide)) and PPG derivatives and PEG-PPG random and preferably block copolymers, poly(hydroxyalkyl) acrylates and methacrylates, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, water soluble polystyrenes including sulfonated polystyrene, hydroxyalkylated and polyhydroxyalkylated polystyrene, and PEG esters and esters of hydroxyalkylated and polyhydroxyalkylated polystyrene; surfactants comprising PEG and hydroperoxides and peroxides of PEG, such as pluronics (e.g. Pluronic-F108 from BASF).
When the polymer comprises a polyalkylene oxide, the polyalkylene oxide moiety can be linear or branched and is preferably a homopolymeric or copolymeric, especially block copolymeric, moiety containing repeat units CnH2nO where n is 2,3 or 4, preferably 2 or 3, especially preferably CH2CH2O, OCHCH3CH2, CH3CHCH2O or CH2CH2CH2O repeat units. Within the PAO moiety, one or more, preferably one or two, of the ether oxygens may be replaced by an amine group NH or NE where E is a bond or an alkyl or hydroxyalkyl group or a (CnH2nO)qExe2x80x2 side chain (where n is 2,3 or 4 and q is an integer, the maximum value for which is set by the molecular weight limit for the PAO and Exe2x80x2 is H or alkyl, a chemical bond or a chromophore).
Any alkyl, alkenyl or alkynyl moieties, unless otherwise defined, preferably have up to 12, especially preferably up to 6 carbons.
In one aspect, a branching group in the backbone of the polyalkylene oxide moiety can be selected from the group consisting of a nitrogen atom and a carbon atom. At least one additional polyalkylene oxidyl group can be attached to the branching group by a chemical bond selected from the group consisting of carbon-carbon, carbon-nitrogen,.and carbon-oxygen chemical bonds, or by a linking group.
Preferred linking groups to a nitrogen branching group include:
methylene groups, [xe2x80x94CH2xe2x80x94];
poly(methylene) groups, [xe2x80x94(CH2)nxe2x80x94] wherein n is an integer from 2 to about 16, such as can be formed by reaction between a nitrogen NH group and an alkylenyl group containing a terminal halide (e.g., Cl, Br, I) or sulfonate group (e.g., methanesulfonate, toluenesulfonate, benzenesulfonate and the like);
alkylenecarbonyl groups [xe2x80x94(CH2)nxe2x80x3xe2x80x94C(xe2x95x90O)xe2x80x94] wherein nxe2x80x3 is an integer from 1 to about 16 such as can be formed by reacting an NH group with a haloalkylenecarbonyl group;
ethylenesulfonylethylene groups [xe2x80x94CH2CH2xe2x80x94S(xe2x95x90O)2xe2x80x94CH2CH2xe2x80x94], such as can be formed by reacting an NH group with a vinylsulfonylethylene group [CH2xe2x95x90CHxe2x80x94S(xe2x95x90O)2xe2x80x94CH2CH2xe2x80x94];
ethylenesulfonylmethyleneoxymethylenesulfonylethylene groups [xe2x80x94CH2CH2xe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94S(xe2x95x90O)2xe2x80x94CH2CH2xe2x80x94], such as can be formed by reacting an NH group with a vinylsulfonylmethyleneoxymethylenesulfonylethylene group [CH2xe2x95x90CHxe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94S(xe2x95x90O)2xe2x80x94CH2CH2xe2x80x94];
ethylenesulfonylmethylenesulfonylethylene groups [xe2x80x94CH2CH2xe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94S(xe2x95x90O)2xe2x80x94CH2CH2xe2x80x94], such as can be formed by reacting an NH branching group with a vinylsulfonylmethylenesulfonylethylene group [CH2xe2x95x90CHxe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94S(xe2x95x90O)2xe2x80x94CH2CH2xe2x80x94];
carbonyl groups [xe2x80x94(Cxe2x95x90O)xe2x80x94] which can comprise an amide linking group formed, for example, by reacting an NH branching group with an activated ester such an N-hydroxysuccinimidyl-ester, or with a mixed anhydride such as a trifluoromethyloxycarbonyl-, or with an acid halide such as an acid chloride, e.g., Clxe2x80x94(Cxe2x95x90O)xe2x80x94;
sulfonyl groups [xe2x80x94S(xe2x95x90O)2xe2x80x94] which can comprise a sulfonamide linking group formed, for example, by reacting an NH branching group with a sulfonyl halide such as a polyalkylene oxidylalkylenesulfonyl chloride, e.g., Clxe2x80x94S(xe2x95x90O)2xe2x80x94(CH2)nxe2x80x94Oxe2x80x94PAO; wherein n is an integer from 2 to about 16 and PAO is a polyalkylene oxidyl group;
carbonyloxy groups [xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94] such as those found in urethane groups such as can be obtained by reacting a polyalkyleneoxy group with phosgene and then with an NH group;
thiocarbonyl groups [xe2x80x94(Cxe2x95x90S)xe2x80x94] such as those found in thiourethane groups such as can be obtained by reacting a polyalkyleneoxy group with thiophosgene and then with an NH group;
alkylenecarbonyloxymethyleneoxycarbonylalkylene groups [xe2x80x94(xe2x80x94CH2xe2x80x94)nxe2x80x2xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C(Rxe2x80x2Rxe2x80x3)xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(xe2x80x94CH2xe2x80x94)nxe2x80x2] where each nxe2x80x2 is independently selected from the group of integers from 1 to 16 and each Rxe2x80x2 and Rxe2x80x3 is independently selected from the group consisting of H and methyl; and,
carbonylalkylenecarbonyl groups [xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)wxe2x80x94C(xe2x95x90O)xe2x80x94] wherein w is an integer from 1 to about 6, such as succinate and adipate.
Preferred linking groups to a carbon branching group include:
ether groups [xe2x80x94Oxe2x80x94];
thioether groups [xe2x80x94Sxe2x80x94];
thiosulfoxide groups [xe2x80x94S(xe2x95x90O)xe2x80x94];
thiosulfonyl groups [xe2x80x94S(xe2x95x90O)2xe2x80x94];
oxycarbonyl groups [xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94];
aminocarbonyl groups [xe2x80x94NHxe2x80x94C(xe2x95x90O)xe2x80x94];
carbonyl groups [xe2x80x94(Cxe2x95x90O)xe2x80x94];
carbonyloxy groups [xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94];
carbonate groups [xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94];
carbonyloxymethyleneoxycarbonylalkylene groups [xe2x80x94(xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C(Rxe2x80x2Rxe2x80x3)xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94(xe2x80x94CH2xe2x80x94)nxe2x80x2xe2x80x94] where nxe2x80x2 is an integer from 1 to 16 and each Rxe2x80x2 and Rxe2x80x3 is independently selected from the group consisting of H and methyl;
urethane groups [xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94]; and
thiourethane groups [xe2x80x94Oxe2x80x94(Cxe2x95x90S)xe2x80x94NHxe2x80x94].
In another aspect, a branching group can comprise the unit xe2x80x94NR1xe2x80x2xe2x80x94CR2xe2x80x2R3xe2x80x2xe2x80x94CR4xe2x80x2R5xe2x80x2xe2x80x94 wherein
R1xe2x80x2 can be selected from the group consisting of H, an alkyl group of from 1 to about 16 carbon atoms which may be linear, branched, saturated, unsaturated, or contain a carbocyclic ring of from 3 to about 10 carbon atoms, or a carbonylalkyl group wherein the alkyl group is defined immediately above;
R2xe2x80x2 and R3xe2x80x2 are independently selected from the group consisting of H, an alkylene group of from 1 to about 16 carbon atoms, which may be linear, branched, saturated or unsaturated, and can contain a carbocyclic ring of from 3 to about 10 carbon atoms and to which is attached a polyalkylene oxidyl group through a heteroatom group selected from the group consisting of NH, O, S, Oxe2x80x94C(xe2x95x90O), and C(xe2x95x90O)xe2x80x94O, e.g., such as 4-(polyalkyleneoxyethylcarbonylaminobutyl), [PAOxe2x80x94CH2CH2C(xe2x95x90O)NHxe2x80x94(CH2)4xe2x80x94, 2-(polyalkyleneoxycarbonyl)ethyl, [PAOxe2x80x94C(xe2x95x90O)CH2CH2xe2x80x94], polyalkyleneoxycarbonylmethyl, [PAOxe2x80x94C(xe2x95x90O)CH2xe2x80x94], polyalkyleneoxyethylaminocarbonylmethyl, [PAOxe2x80x94CH2CH2NHC(xe2x95x90O)CH2xe2x80x94], polyalkyleneoxyethylaminocarbonylethyl, [PAOxe2x80x94CH2CH2NHC(xe2x95x90O)CH2CH2xe2x80x94], polyalkyleneoxymethyl, [C], and polyalkyleneoxyethylthiomethyl, [PAOxe2x80x94CH2CH2xe2x80x94Sxe2x80x94CH2xe2x80x94];
R4xe2x80x2 and R5xe2x80x2 are independently selected from the group consisting of H, an alkyl group of from 1 to about 16 carbon atoms which may be linear, branched, saturated, unsaturated, or contain a carbocyclic ring of from 3 to about 10 carbon atoms, or a carbonylalkyl group wherein the alkyl group is defined above, or, preferably, where both R4xe2x80x2 and R5xe2x80x2 are taken together form a carbonyl group;
and wherein at least one of R2xe2x80x2R3xe2x80x2 is not H.
Preferred units xe2x80x94NR1xe2x80x2xe2x80x94CR2xe2x80x2R3xe2x80x2xe2x80x94CR4xe2x80x2R5xe2x80x2xe2x80x94 are selected from the group consisting of lysine, aspartic acid, glutamic acid, cysteine, and serine in the backbone of the polyalkylene oxide moiety and contain least one additional polyalkylene oxide attached, for example, to the epsilon amine site of lysine, to the gamma carboxylic acid site of aspartic acid, to the delta carboxylic acid site of glutamic acid, to the beta sulfhydryl group in cysteine, and to the beta hydroxy site of serine.
In another aspect, one branching group and a carbon atom in the backbone of the polyalkylene oxide moiety or two branching groups in the backbone of the polyalkylene oxide moiety can be joined by an alkylene group of from 2 to 12 carbon atoms. The alkylene group can be linear or branched such as ethylene, propylene, butylene, isobutylene, pentylene, hexylene, octylene, decylene, and dodecylene. The alkylene group can be saturated or unsaturated such as 2-butenylidene, isoprenylene, and 2-butynylidene. In another aspect, the alkylene group can comprise a saturated or unsaturated cyclic group such as cyclopropylidene, cyclobutylidene, 1,2-cyclopentylidene, 1,3-cyclopentylidene, 1,2-cyclohexylidene, 1,3-cyclohexylidene, 1,4-cyclohexylidene, a cyclohexenylidene ring such as can be formed by a Diels-Alder reaction between a diene and a dieneophile, 1,4-cycloheylidenebismethylene, ethylene-1,2-cyclopropylidenemethylene, 1,1-spirocycloproylidene-bismethylene, and the like, and which can contain an oxygen or sulfur ether atom, such as a 2,5-tetrahydrofuranylene group and a 2,6-tetrahydro-pyranylene group.
In another aspect, one branching group and a carbon atom in the backbone of the polyalkylene oxide moiety or two branching groups in the backbone of the polyalkylene oxide moiety can be separated by an aromatic ring of 6 to 14 carbon atoms such as p-phenylene, or m-phenylene, or m-toluidene, 9,10-anthracenylidene, or 1,4-naphthalenylidene, or an aralkylene group such as p-phenylenebismethylene, or 9,10-anthracenylidene-bismethylene, and which aromatic ring can comprise a 5- or 6-membered heterocyclylene group containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur such a 2,6-pyridinylene, 1,4-imidazolidene, 5,8-quinolinylidene, and 1,1-spiro-2,6-dithiacyclohexylene, or a symmetrical triazinylene group.
The polymeric compounds may be homo- or copolymers, and where copolymers may be random, block or graft, and may contain individual comonomer residues such as the diamine residues in the poloxamine polymers. Poly(alkylene oxide) polymers are especially preferred.
Such poly(alkylene oxide) compounds are readily available commercially, e.g. as Pluronics, Tetronics or PEGs of various molecular weights.
The poly(alkylene oxides) may contain a poly(alkylene oxidyl) moiety (e.g. a moiety of formula ((X)nO)p, where X is an alkylene group (e.g. a C2-4 alkylene) and n and p are positive integers (n conveniently being 1 to 5 and p from 1 to 20000), optionally incorporating an alkylene-amino or alkylenediamino group such as an ethyleneamine or ethyienediamine group) and may consist simply of such a moiety terminated by simple functional groups, e.g. hydroxyl, amine, sulphate carboxylate, phosphate and phosphonate groups.
Conjugates of the polymers used according to the invention may contain moieties, other than the water soluble (i.e. hydrophilic) polymer moiety, covalently bonded together, e.g. chromophores, lipophilic groups (e.g. phospholipid groups), biotargetting vector groups, and groups detectable in in vivo diagnostic imaging modalities such as for example MR and X-ray (e.g. CT) imaging.
Preformed encapsulated gas bubbles are especially preferred as contrast agents for sonoluminescence imaging. Similar gas-filled bubbles are useful as contrast agents for ultrasound imaging. The enclosed gas may be air, xenon, argon, helium or any other physiologically acceptable gas. Mixtures of gases such as xenon and nitrogen, argon and nitrogen, and helium and nitrogen are also within the scope of the invention. A preferable mixture is 1 to 99% xenon in nitrogen. Especially preferable is a mixture of 1 to 10% xenon in nitrogen.
Particular examples of materials suitable for use as contrast agents for sonoluminescence imaging in the present invention are: particulate suspensions containing 1,1xe2x80x2,3,3,3xe2x80x2,3xe2x80x2-hexamethylindotricarbocyanine iodide, prepared as described in Example 1 of WO 97/13490; and the compounds of Examples 1-7 of WO 96/17628; all of which Examples are incorporated herein by reference.
In the methods of the invention, the contrast agent is administered to the subject (e.g. a human patient) under investigation in a manner such that it may reach the target zone of interest, e.g. the breast, brain, liver, lymph nodes, skin lesion etc. Such administration may be by any conventional route, e.g. administration into the gastrointestinal tract, injection or infusion into the vasculature, subcutaneous, intramuscular or interstitial injection or infusion, sublingual or nasal administration, administration into the lungs, vagina, bladder or uterus, topical or transdermal application, etc. In general, injection or infusion into the vasculature, subcutaneous or interstitial administration, topical application or administration into the gastrointestinal tract will be preferred. Administration may take place at the target zone or at a site from which the contrast agent is transported to the target site, e.g. by uptake through the walls of the gastrointestinal tract or by transport within the blood vessels. Information recording (e.g. image generation) may take place before the contrast agent has reached the target zone but in that event will also take place after the contrast agent has reached the target site.
For the information generation procedure, the target zone is irradiated with ultrasound, preferably focused ultrasound, e.g. of frequency 0.01 to 10 MHz, especially 0.1 to 3 MHz, particularly 0.5 to 2.5 MHz. The ultrasound irradiation may be continuous and uniform at the target zone or more preferably may be frequency- and/or amplitude-modulated. One form of amplitude modulation that may be used is pulsed ultrasound. In general however frequency and amplitude modulation will involve imposition of a variation in frequency and/or amplitude which has its own characteristic frequency (the ultrasound modulation frequency). The modulation frequency will be between 0 and 100 kHz, especially 0 and 30 kHz, particularly 0 and 10 kHz.
Where the contrast agent is sonoluminescent and the information is generated using detected sonoluminescent emissions, the ultrasound is preferably modulated, especially amplitude-modulated. Particularly preferably the amplitude minima are below the level where resulting sonoluminescence is detectable while the amplitude maxima are above that level. In this case the generated light will be modulated at the modulation frequency and harmonics thereof. Detection of the amplitude of the light at one or more of those frequencies will facilitate separation from the background of light from ambient sources. Where spatial resolution is not critical, and where background noise is reduced or eliminated (e.g. by conducting the procedure in the dark or in visible light filtered to remove red to near infra-red components) it may not be necessary to extract a modulated component from the detected light signal.
Where the frequency of the sonoluminescent emission is dependent on the microenvironment of the contrast agent, the detected light may be filtered or frequency analysed to separate out the components generated under different conditions, e.g. of pH or oxygen concentration, and so provide information about the physicochemical nature of the target zone.
Where the detected light is sonoluminescence, it is especially preferred that the ultrasound irradiation be focused to ensure that the detected signal derives from a clearly locatable target zone. For this reason, the use of the quasi-ballistic component of the sonoluminescence (i.e. that component which follows a relatively straight path from the light generation site to the first part of the light detector assembly it meets) may also be preferred.
Where sonoluminescence generation is dependent on the frequency of the ultrasound irradiation, modulation of the ultrasound frequency may be used in the same way as modulation of ultrasound amplitude to select a relatively low noise component of the detected light signal from which to generate the desired information or image.
In embodiments of the invention where the contrast agent is not sonoluminescent, the target zone will also be illuminated with light of a frequency in the range 300 to 1300 nm, preferably 600 to 1300 nm especially 650 to 900 nm. Such light may be polychromatic and polydirectional; however, preferably one will use a substantially monochromatic focused light beam, especially preferably a laser beam, e.g. from a tunable dye laser. The incident light may again be modulated in amplitude and/or frequency, with amplitude modulation being preferred. As with the ultrasound modulation discussed above, modulation of the incident light beam at a characteristic modulation frequency and analysis of the detected light signal and extraction of a component also modulated at that characteristic modulation frequency allows generation of information and images using a signal containing lower level of noise.
Where the interaction of contrast agent and illuminating light is a scattering interaction, e.g. where the contrast agent is a chromophore-free particulate, the detected light signal may be frequency analysed to extract the component which is Doppler shifted by interaction with ultrasound in the target zone, thereby again reducing the noise level in the component used for information generation. Similarly, if desired, the quasi-ballistic component of the detected amplitude (or frequency) modulated incident light may be used for information generation.
Where in the methods of the invention the contrast agent emits light or scatters light, it is particularly preferred to detect the quasi-ballistic component of the light from the target zone. Where the target zone is illuminated, the presence of a contrast agent may reduce the quasi-ballistic component of the transmitted irradiating light, i.e. the quasi-ballistic component along the optical axis between light source and light detector. However, away from that axis the quasi-ballistic component of light originating from or scattered in the target zone will be increased.
In the method of the invention, ultrasound irradiation is preferably effected using a focused transducer, e.g. a 3.5 MHz transducer with a focal length of 12.5 cm operating with a driving electrical signal at 3.5 MHz consisting of 50 xcexcsec pulses with peak voltages of 100 volts and a repetition rate of 40 Hz. Generally, the ultrasound frequency can be between 0.1 and 10 MHz and the focal length should be between 1 and 20 cm. The peak pressure at the focus should be less than 50 bars. Any laser illumination is preferably at a power level of 10 milliwatts or less. Light detection may be effected using conventional photodetectors, preferably a photomultiplier tube such as the Hamamatsu HC. 123-01. To minimise xe2x80x9cnoisexe2x80x9d, the detections may be shielded to prevent light other than from the subject under study from reaching the photodetectors.
Ultrasound irradiation and light irradiation and detection may be effected using devices located externally of the subject under study or alternatively using devices inserted into a body cavity through a natural or surgically produced orifice.
All of the publications referred to herein are incorporated herein by reference.
The contrast agents of the invention may be administered to patients for imaging in amounts sufficient to be effective in the particular technique.
The dosage of the contrast agents will depend on the site being investigated, the nature of the contrast agent and the details of the imaging procedure. For intravenous feeding with emulsions, doses up to 1000 mg/kg/day have generally proven safe, but for imaging the doses will generally be 0.1 to 10 ml/kg of the suspension or 15 to 1500 mg/kg of the oil component of the suspension. For suspended solids the dose will vary from 100 to 500 mg/kg of the solid. For polymers the dose will vary from 1 to 700 mg/kg.
The contrast agents may be formulated with conventional pharmaceutical or veterinary aids, for example emulsifiers, fatty acid esters, gelling agents, stabilizers, antioxidants, osmolality adjusting agents, buffers, pH adjusting agents, etc., and may be in a form suitable for parenteral or enteral administration, for example injection or infusion or administration directly into a body cavity having an external escape duct, for example the gastrointestinal tract, the bladder or the uterus. Thus the contrast agents may be presented in conventional pharmaceutical administration forms such as tablets, capsules, powders, solutions, suspensions, dispersions, syrups, suppositories etc. However, solutions, suspensions and dispersions in physiologically acceptable carrier media, for example water for injections, will generally be preferred.
The contrast agents may therefore be formulated for administration using physiologically acceptable carriers or excipients in a manner fully within the skill of the art. For example, the contrast agents, optionally with the addition of pharmaceutically acceptable excipients, may be suspended or dissolved in an aqueous medium, with the resulting solution or suspension then being sterilized.
For some portions of the body, the most preferred mode for administering the contrast agents is parenteral, e.g. intravenous administration. Parenterally administrable forms, e.g. intravenous solutions or dispersions, should be sterile and free from physiologically unacceptable agents, and should have low osmolality to minimize irritation or other adverse effects upon administration, and thus the contrast medium should preferably be isotonic or slightly hypertonic. Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions or dispersions such as Sodium Chloride Injection, Ringer""s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer""s Injection and other solutions such as are described in Remington""s Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975). The solutions or dispersions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the dyes and which will not interfere with manufacture, storage or use.
The invention will now be described further with reference to the following non-limiting Examples.